• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.247-250
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• 2002

본 연구에서는 형질전환된 P. pastoris를 이용한 재조합 HBsAg 생산에서 유가식 배양시 공급배치 탄소원으로 sorbitol이 단백질 발현에 미치는 영향을 glycerol과 비교하여 실험하였다. 유가식 배양 공급배지 탄소원으로 50% sorbitol을 이용했을 때 50% glycerol을 이용하는 경우보다 세포 증식 측면에서는 methanal 유도 후 균체 농도가 낮은 경향을 보였으나 이것은 glycerol 이 sorbitol 보다 에너지원으로써 높은 affinity를 가지기 때문인 것으로 판단된다. 하지만 단위 건조 균체량당 단백질 발현량은 50% sorbitol을 공급 한 경우 50% glycerol을 공급한 경우 보다 12% 향상되는 결과를 보였다. 따라서 유가식 배양용 공급 탄소원으로 sorbitol을 이용했을 때 glycerol을 이용하는 것보다 AOX promoter에 의한 단백질 발현에 보다 긍적적인 효과가 있는 것을 확인 할 수 있었다.

• KSBB Journal
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• 제21권4호
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• pp.260-266
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• 2006

본 연구에서는 P. pastoris를 이용한 유전자 재조합 HBsAg 생산에서 메탄올 유도시 여러 가지 첨가물들의 영향에 대해서 알아보았다. 회분식 배양에서 탄소원으로 글리세롤을 사용하다가 유가식 배양의 공급 탄소원으로 글리세롤이 아닌 당알콜인 sorbitol로 대체하였을 때 단백질 발현이 향상된 결과를 보였다. 또한 메탄올 유도시에 적당량의 아미노산 혼합물 첨가는 세포증식에는 영향이 없었지만 단백질 발현율은 크게 증가시켰다. 계면활성제인 Trition X-100의 첨가는 세포증식과 단백질 발현을 현저히 감소시켰지만, Pluronic F-68를 첨가했을 경우 세포증식의 저해영향 없이 단백질 발현율을 향상시켰다. 배지 부피의 0.01%(v/v)으로 oleic acid를 첨가하면 플라스크 배양에서는 단백질 발현에 긍정적인 효과를 보였으나, 5 L 발효조 배양에서는 메탄올 유도 시간이 지속되면서 첨가하지 않은 경우에 비해 발현율이 낮아지는 결과를 보였다. 마지막으로 trace salts는 첨가량에 따라 세포증식에는 영향이 없으며 단백질 발현에는 소량 trace salts 첨가로 단백질 발현에 긍정적인 효과를 보였다. 하지만 첨가량이 많아질수록 단백질 발현에는 부정적인 영향을 보임을 확인할 수 있었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.191-194
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• 2003

본 연구에서는 산업적으로 중요성이 날로 증가하고 있는 PLC의 대량 생산을 위해서 B. cereus ATCC10987에서 분리된 PLC 유전자를 pPICZa C에 삽입한 P. pastoris system을 개발하여 extracellular PLC를 생산하였다. 또한, 공업적 이용을 위해 여러 조건에서 B. cereus와 P. pastoris에서 생산된 extracellular PLC의 활성도를 측정하여 특성을 살펴보았다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.233-235
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• 2003

본 연구에서는 산업적으로 중요성이 날로 증가하고 있는 PLC의 대량 생산을 위해서 B. cereus ATCC10987에서 분리된 PLC 유전자를 $pPICZ{\alpha}$ C에 삽입한 P. pastoris system을 개발하여 extracellular PLC를 생산하였다. 또한, 여러 조건에서 B. cereus와 P. pastoris에서 생산된 extracellular PLC의 활성도를 측정하여 특성을 살펴보았다.

• 한국미생물·생명공학회지
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• 제38권1호
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• pp.40-45
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• 2010

Agarose의 $\beta$-1,4결함을 분해하는 Zobellia galactanivorans 유래의 $\beta$-agarase 유전자(agaB)는 클로닝 되었고, AOX1(alcohol oxidase 1, methanol inducible) promoter 하류에 Saccharomyces cerevisiae mating factor alpha-1 secretion signal($MF{\alpha}1$)를 연결하여 $MF{\alpha}1$-AgaB를 구축하였다. 구축된 plasmid pPIC-AgaB(9 kb)를 Pichia pastoris genome에 HIS4 gene 위치에 integration하였고, colony PCR을 통해 확인하였다. Methanol 첨가 배지에서 자란 형질전환체는 iodine solution의 첨가에 의해 red halos를 보였으며, P.pastoris에서 agaB의 효율적 분비 발현을 확인하였다. SDS-PAGE와 zymographic analysis에서 $\beta$-agarase의 분자량은 약 53 kDa으로 추정되었으며, 15% 정도의 N-linked glycosylation이 일어났음을 알 수 있었다. P.pastoris GS115/pPIC-AgaB의 48시간 baffled flask culture에서 세포외 $\beta$-agarase의 활성은 각각 0.1, 0.5, 1% methanol의 유도에 의해 1.34, 1.42 그리고 1.53 units/mL의 활성을 보였다. 대부분의 $\beta$-agarase의 활성은 세포 외에서 관찰되었고, 분비효율은 98%였으며 분비시의 glycosylation에 의해 열안정성도 증가되었다.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.915-921
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• 2001

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. In order to express a high level of fungal phytase in Saccharomyces cerevisiae, various expression vectors were constructed with different combinations of promoters, translation enhancers, signal peptides, and terminator. Three different promoters fused to the phytase gene (phyA) from Aspergillus niger were tested: a galactokinase (GAL1) promoter, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, and yeast hybrid ADH2-GPD promoter consisting of alcohol dehydrogenase II (ADH2) and a GPD promoter. The signal peptides of phytase, glucose oxidase (GO), and rice amylase 1A(RAmy1A) were included. Plus, the translation enhancers of the ${\Omega}$ sequence and UTR70 from the tobacco mosaic virus (TMV) and spinach, respectively, were also tested. Among the recombinant vectors, pGphyA06 containing the GPD promoter, the ${\Omega}$ sequence, RAmy1A, and GAL7 terminator expressed the highest phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase was also performed by inserting an endoplasmic reticulum (ER) retention signal, KDEL sequence, into the C-terminus of the phytase within the vector pHphyA-6. It appeared that the KDEL sequence directed most of the early expression of phytase into the intracellular compartment yet more than $60\%$ of the total phytase activity was still retained within the cell even after the prolonged (>3 days) incubation of the transformant. However, the intracellular enzyme activity of the transformant without a KDEL sequence was as high as that of the extracellular one, thereby strongly suggesting that the secretion of phytase in S. cerevisiae appeared to be the rate-limiting step for the expression of a large amount of extracellular recombinant phytase, when compared with other yeasts.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2016년도 춘계학술대회 및 임시총회
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• pp.27-27
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• 2016

The ascomycete fungus Fusarium graminearum is the most common pathogen of Fusarium head blight (FHB), a devastating disease for major cereal crops worldwide. FHB causes significant crop losses by reducing grain yield and quality as well as contaminating cereals with trichothecenes and zearalenone (ZEA) that pose a serious threat to animal health and food safety. ZEA is a causative agent of hyperestrogenic syndrome in mammals and can result in reproductive disorders in farm animals. In F. graminearum, the ZEA biosynthetic cluster is composed of four genes, PKS4, PKS13, ZEB1, and ZEB2, which encode a reducing polyketide synthase, a nonreducing polyketide synthase, an isoamyl alcohol oxidase, and a transcription factor, respectively. Although it is known that ZEB2 primarily acts as a regulator of ZEA biosynthetic cluster genes, the mechanism underlying this regulation remains undetermined. In this study, two isoforms (ZEB2L and ZEB2S) from the ZEB2 gene in F. graminearum were characterized. It was revealed that ZEB2L contains a basic leucine zipper (bZIP) DNA-binding domain at the N-terminus, whereas ZEB2S is an N-terminally truncated form of ZEB2L that lacks the bZIP domain. Interestingly, ZEA triggered the induction of both ZEB2L and ZEB2S transcription. In ZEA producing condition, the expression of ZEB2S transcripts via alternative promoter usage was directly or indirectly initiated by ZEA. Physical interaction between ZEB2L and ZEB2L as well as between ZEB2L and ZEB2S was observed in the nucleus. The ZEB2S-ZEB2S interaction was detected in both the cytosol and the nucleus. ZEB2L-ZEB2L oligomers activated ZEA biosynthetic cluster genes, including ZEB2L. ZEB2S inhibited ZEB2L transcription by forming ZEB2L-ZEB2S heterodimers, which reduced the DNA-binding activity of ZEB2L. This study provides insight into the autoregulation of ZEB2 expression by alternative promoter usage and a feedback loop during ZEA production.

• BMB Reports
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• 제38권3호
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• pp.294-299
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• 2005

In this report, we describe the high-yield secretory expression of the recombinant human anti-HBsAg Fab fragment from Pichia pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of alcohol oxidase promoter) into the genome of the yeast cells. The fed-batch fermentations were carried out in a 5 L scale. Both chains of the Fab were successfully expressed upon methanol induction. The absorbance ($OD_{600}$) of the broth can reach 350~500 at the end of fed-batch phase. After the induction, the expression level of the recombinant Fab (soluble) reached 420~458 mg/L. The recombinant Fab fragment was purified from the crude culture supernatant by ion exchange chromatography and the purity of the recombinant Fab fragment was over 95%. The affinity activities of the crude fermentation supernatant and the purified Fab were analyzed by indirect ELISA, which showed that the purified recombinant Fab fragment had high affinity activity with hepatitis B surface antigen.

• BMB Reports
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• 제35권5호
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• pp.476-481
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• 2002

The CHH/MIH/GIH peptide family of black tiger prawn (Paneaus monodon) is important in shrimp reproduction and growth enhancement. In this study, the cDNA that encodes the complete peptide that is related to the CHH/MIH/GIH family (so-called, Pem-CMG) in the eyestalk of P. monodon was successfully expressed in a methylotrophic yeast Pichia pastoris under the control of an alcohol oxidase promoter. In order to obtain the secreted Pem-CMG, a secretion signal of either the Saccharomyces cerevisiae $\alpha$-factor or Pem-CMG was employed. The results demonstrated that ${\alpha}Pem$-CMG, either with (${\alpha}2EACMG$) or without (${\alpha}CMG$) the Glu-Ala repeats, was secreted into the medium, while Pem-CMG with its own secretion signal failed to be secreted. The total protein amount that was secreted from the transformant that contained either ${\alpha}2EACMG$ or ${\alpha}CMG$ was approximately 60 mg/l and 150 mg/l, respectively. The N-terminus of the Pem-CMG peptide of both ${\alpha}2EACMG$ and ${\alpha}CMG$ was correctly processed. This produced the mature Pem-CMG peptide.

• Journal of Microbiology and Biotechnology
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• 제26권3호
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• pp.461-468
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• 2016

In recent years, various naturally occurring defence peptides such as plectasin have attracted considerable research interest because they could serve as alternatives to antibiotics. However, the production of plectasin from natural microorganisms is still not commercially feasible because of its low expression levels and weak stability. A tandemly arrayed plectasin gene (1,002 bp) from Pseudoplectania nigrella was generated using the isoschizomer construction method, and was inserted into the pPICZαA vector and expressed in Pichia pastoris. The selected P. pastoris strain yielded 143 μg/ml recombinant plectasin (Ple) under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter. Ple was estimated by SDS-PAGE to be 41 kDa. In vitro studies have shown that Ple efficiently inhibited the growth of several gram-positive bacteria such as Streptococcus suis and Staphylococcus aureus. S. suis is the most sensitive bacterial species to Ple, with a minimum inhibitory concentration (MIC) of 4 μg/ml. Importantly, Ple exhibited resistance to pepsin but it was quite sensitive to trypsin and maintained antimicrobial activity over a wide pH range (pH 2.0 to 10.0). P. pastoris offers an attractive system for the cost-effective production of Ple. The antimicrobial activity of Ple suggested that it could be a potential alternative to antibiotics against S. suis and S. aureus infections.